Protein synthesis by the ribosome is central to life and disease. Biochemical, kinetic and structural studies have provided detailed views of the intermediates and mechanisms in translation. To complement these approaches, we developed single-molecule fluorescence methods to track both conformational and compositional dynamics of the translation machinery in real time. Here we build on our methodological developments over the past funding period, and leverage the strengths of single-molecule methods watch translation in real time. In Aim 1 we focus on basic steps of translation: the timing of initiation events, focusing on IF1 and IF3 and initiator tRNA, termination driven by RF1, 2 and 3 and recycling by RRF and EF-G. We will use conformational and compositional signals to track both simultaneously in real time using novel instrumentation. In Aim 2, we will shift to investigate rar pausing, stalling and reading- frame changes that occur during elongation due to mRNA sequence or structure. We will study the role of SD interactions in reading frame, and the influence of RNA structure on elongation rates. We will then study the interplay of these RNA features that lead to -1 and +1 frame shifting and ribosome hopping, using single-molecule fluorescence to track pathways, branch points and mechanism. In Aim 3, we examine factors that interact in the peptide exit tunnel to cause pausing and stalling, including ribosomal nascent chains and antibiotics such as macrolides. We will follow the evolution of stalling for several systems, including SecM, TnaC, and ErmCL, confirming the sequence features of the nascent chain required for stalling and their contributions to the kinetic pathway to the stalled or paused state. For ErmCL, we will determine the interplay of erythromycin and nascent chain that causes stalling, and correlate the stall behavior with drug occupancy. Finally, we will investigate generally the dynamics of macrolide binding to ribosomes, and how drug occupancy and dynamics change as a function of elongation for both sensitive and resistant ribosomes. The proposed research is buttressed by strong collaborations to support bulk kinetic analysis, reagent preparation, and in vivo correlation. The expected results should provide a dynamic overview of initiation, termination, and elongation, and how general dynamics are perturbed to change protein expression.